Fig 2: Fibrosis-related and apoptosis-related proteins in 3T3 and LX-2 cells measured with Western blotting. (A) Relative protein level of p53, cleaved caspase 3 (casp 3), tBid, DR4, DR5, uPA and PAI-1 normalized to GAPDH. (B) Relative protein level of ß-crystallin, a-SMA, collagen I and collagen III normalized to GAPDH. (C) Representative blots for each protein measured with Western blotting from triplicate results. +P < 0.05, ++P < 0.01, compared with SSL; *P < 0.05, **P < 0.01, compared with TRAIL; #P < 0.05, ##P < 0.01, compared with SSL-TRAIL

Fig 3: Clusterin supplementation attenuates TGFß2-induced fibrogenic process. Serum-starved HTM cells treated with TGFß2 exhibited significant increase in COL1A (WCL), TSP1 (WCL and CM), and PAI1 (CM) which was significantly suppressed by supplementation of rhCLU. ß-actin or GAPDH was used as a loading control for WCL, and ponceau-S band was used as a loading control for CM. Values represent the mean ± SEM, where n = 4 (biological replicates). * p = 0.05 was considered significant. CM, conditioned media; SEM, standard error of mean; WCL, whole-cell lysate

Fig 4: Single-cell RNA sequencing reveals heterogeneity and distinct subsets of fibroblasts.a Diagram depicting the generation of tdTomatorPßC mouse and PDGFRß+ cell-specific expression of tdTomato from 8-week old and their analyses at 10-week old. b Illustration of droplet-based single-cell RNA sequencing (scRNA-seq) of PDGFRß+tdTomato+ IntSCs sorted from the small intestine of tdTomatorPßC mice. c Visualization of unsupervised clustering of 7 distinct PDGFRß+ IntSC clusters by Uniform manifold approximation and projection (UMAP) in the small intestine of tdTomatorPßC mice. vFB, intestinal villi fibroblast; FB, fibroblast; SMC, smooth muscle cell; MC, mural cell. d Heatmap displaying the scaled expression patterns of top ten differentially expressed genes for random sampled cells (maximum thousand cells) for each indicated clusters. e List of representative marker genes of each of the seven PDGFRß+ IntSC clusters (left) and violin plots (right) showing the expression of top-ranking marker genes for each cluster. Log normalized read counts as y-axis (normalized expression). f UMAP visualization of unsupervised clustering of seven distinct PDGFRß+ IntSC clusters in the small intestine of Lats1/2i?-tdTomatorPßC mice. g Gene expression levels of Vegfc and Vegfa in tdTomatorPßC and Lats1/2i?-tdTomatorPßC mice projected on UMAP plot. Note that specific subsets of PDGFRß+ IntSCs (vFB1-3) in the small intestine of Lats1/2i?-tdTomatorPßC mice show higher expression of Vegfc compared with those of tdTomatorPßC mice. h, i Representative images of PDGFRß+ IntSCs in WT mouse reveal expressions of each fibroblast-specific markers: PAI-1+ vFB1, Serpina3n+ vFB2, P2X1+ vFB3, Ackr4+ or Grem1+ FB4, and PDGFRa+ Sox6+ FB5. Each white box in the lower left corner is a magnified view. White asterisks indicate lacteals and white arrowheads indicate each fibroblast cluster-specific cell type stained with the indicated marker. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 20 µm. j Schematic images depicting the anatomic distribution of indicated markers for vFB1-3, FB4, FB5, SMC, MC, capillary plexus, and lacteal in intestinal villi of adult WT mouse. vFB1-3 are uniformly distributed around the lacteal, whereas FB4 is mainly located in the submucosal area and FB5 is mostly placed under the intestinal epithelium. Black dashed boxes are magnified in the right panels.

Fig 5: Immunoblots of PAI-1 in patients with diabetic nephropathy. The total protein was harvested, separated on 10% SDS-PAGE gels and subjected to immunoblot analysis. The primary antibodies against PAI-1 (~50 kDa) and ß-actin were anti-PAI-1 (dilution, 1:200) and anti-ß-actin (dilution, 1:10,000), respectively. The secondary antibody used in this study was goat anti-mouse IgG-HRP (dilution, 1:10,000). Bound antibodies were detected using an ECL system. (A) Representative blots. (B) The mean normalized OD of PAI-1 protein bands relative to the OD of ß-actin bands from each of the 4 groups. PAI-1, plasminogen activator inhibitor-1; OD, optical density.